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Inactivated enzymes as probes of the structure of arabinoxylans as observed by atomic force microscopy

Lookup NU author(s): Emeritus Professor Harry Gilbert


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The complex structures of water-soluble wheat arabinoxylans have been mapped along individual molecules, and within populations, using the visualisation of the binding of inactivated enzymes by atomic force microscopy (AFM). It was demonstrated that site-directed mutagenesis (SDM) can be used to produce inactive enzymes as structural probes. For the SDM mutants AFM has been used to compare the binding of different xylanases to arabinoxylans. Xylanase mutant E386A, derived from the Xyn11A enzyme (Neocallimastrix patriciarium), was shown to bind randomly along arabinoxylan molecules. The xylanase binding was also monitored following Aspergillus niger arabinofuranosidase pre-treatment of samples. It was demonstrated that removal of arabinose side chains significantly altered the binding pattern of the inactivated enzyme. Xylanase mutant E246A, derived from the Xyn10A enzyme (Cellvibrio japonicus), was found to show deviations from random binding to the arabinoxylan chains. It is believed that this is due to the effect of a small residual catalytic activity of the enzyme that alters the binding pattern of the probe. Control procedures were developed and assessed to establish that the interactions between the modified xylanases and the arabinoxylans were specific interactions. The experimental data demonstrates the potential for using inactivated enzymes and AFM to probe the structural heterogeneity of individual polysaccharide molecules. © 2003 Elsevier Ltd. All rights reserved.

Publication metadata

Author(s): Adams EL, Kroon PA, Williamson G, Gilbert HJ, Morris VJ

Publication type: Article

Publication status: Published

Journal: Carbohydrate Research

Year: 2004

Volume: 339

Issue: 3

Pages: 579-590

ISSN (print): 0008-6215

ISSN (electronic): 1873-426X

Publisher: Pergamon


DOI: 10.1016/j.carres.2003.11.023

PubMed id: 15013394


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